The therapeutic mavericks: Potent immunomodulating chaperones capable of treating human diseases

Abstract Two major chaperones, calreticulin (CRT) and binding immunoglobulin protein (GRP78/BiP) dependent on their location, have immunoregulatory or anti‐inflammatory functions respectively. CRT induces pro‐inflammatory cytokines, dendritic cell (DC) maturation and activates cytotoxic T cells against tumours. By contrast, GRP78/BiP induces anti‐inflammatory cytokines, inhibits DC maturation and heightens T‐regulatory cell responses. These latter functions rebalance immune homeostasis in inflammatory diseases, such as rheumatoid arthritis. Both chaperones are therapeutically relevant agents acting primarily on monocytes/DCs. Endogenous exposure of CRT on cancer cell surfaces acts as an ‘eat‐me’ signal and facilitates improved elimination of stressed and dying tumour cells by DCs. Therefore, therapeutics that promote endogenous CRT translocation to the cell surface can improve the removal of cancer cells. However, infused recombinant CRT dampens this cancer cell eradication by binding directly to the DCs. Low levels of endogenous BiP appear as a surface biomarker of endoplasmic reticulum (ER) stress in some types of tumour cells, a reflection of cells undergoing proliferation, in which resulting hypoxia and nutrient deprivation perturb ER homeostasis triggering the unfolded protein response, leading to increased expression of GRP78/BiP and altered cellular location. Conversely, infusion of an analogue of GRP78/BiP (IRL201805) can lead to long‐term immune resetting and restoration of immune homeostasis. The therapeutic potential of both chaperones relies on them being relocated from their intracellular ER environment. Ongoing clinical trials are employing therapeutic interventions to either enhance endogenous cell surface CRT or infuse IRL201805, thereby triggering several disease‐relevant immune responses leading to a beneficial clinical outcome.


| INTRODUC TI ON
All organisms require a quality control system to assist in protein holding, folding, modification, degradation and for transportation of mature proteins to other organelles or secretion from the cell.
The chaperones are a group of proteins that fulfil these roles and ultimately prevent nonspecific aggregation of unfolded proteins.
Many molecular chaperones are also termed heat shock proteins (HSPs); they were first discovered in Drosophila and bacteria 1 and become abundant under some environmentally stressful conditions, such as higher than normal temperature, pH or glucose fluctuation and hypoxic conditions. These conditions are prevalent in many diseases where there is infection, chronic inflammation, solid tumour growth or metabolic imbalance. In such harsh conditions, it is important that essential proteins are produced and correctly folded for the survival of the organism. In humans, most chaperones are believed to be located at some time in the ER and more infrequently in the cytosol, nucleus, lysosomes and mitochondria, 2 where they have numerous and distinct functions. Chaperones have historically been allotted into HSP families based on their molecular weight, for example HSP10, HSP27, HSP40, HSP60, HSP70, HSP90, HSP110 and chaperonins. 2 In the ER, GRP78/BiP (a HSP70 family member) is an ATP-dependent chaperone, while calreticulin (gene name: CALR or protein name: CRT) is a Ca 2+ dependent lectin chaperone (Figure 1). Common dogmas imply that chaperones are located and exclusively function solely within cells. But we and others have found that some chaperones including GRP78/BiP [3][4][5][6][7] and CRT have immune-modulating functions outside of the cell. [8][9][10] Moreover, whereas CRT protein expression changes in response to raised cell temperature, GRP78/BiP is not particularly responsive to heat shock.
Eventual release of chaperones into the extracellular environment can occur either in a highly regulated manner or simply during cell necrosis. Individually, extracellular CRT [11][12][13][14][15][16] and GRP78/BiP [17][18][19] have been monitored as biomarkers on the cell surface or in the serum/plasma, or other fluids of the body of patients with various diseases (see Table 1). Once outside the cell, CRT 11 and GRP78/BiP 20 have both been detected in healthy human plasma/serum at low nanogram/ml levels and often at higher concentrations in plasma/ serum concentrations in various diseases. However, central to understanding of these chaperones is that their increased expression occurs because of disease pathology and not the cause. What is evident is that CRT and GRP78/BiP appear to provoke or restrain immune responses to diseases, once in the extracellular environment. 21 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17], N-domain (aa , a hair-pin looped P-domain (aa 198-308) and C-domain (aa 309-417). The protein structure prediction of CRT presented is the AlphaFold2 model for CRT (AF-P27797-F1) (http://marrv el.org/human/ gene/811). (B) The GRP78/BiP protein structure was prepared by PyMOL software V2.5.1 (PDB 5E84), the nucleotide-binding domain is in the lower region of the protein model, while the substrate-binding domain is situated towards the upper part of the molecule. TA B L E 1 Cell surface/extracellular CRT or GRP78/BiP associated with human disease recognition molecule that can promote pro-inflammatory immune changes, whereas GRP78/BiP does not fit this paradigm and has been defined as 'regulatory-associated molecular pattern (RAMP)' that in the extracellular environment can act to promote immediate anti-inflammatory and pro-resolution signals, and thus both chaperones can be exploited for therapeutic use.

| Chaperone history and evolution
Interestingly, chaperone function spawned from a dogma that proteins folded spontaneously requiring little help from other proteins.
In 1962, Anfinsen describing his 'thermodynamic hypothesis' concluded that the native form of a protein is its most thermodynamically stable configuration and side-chain functional groups influenced the correct folding of proteins by the formation of intramolecular disulphide linkages. 22 Later, it became apparent that a group of highly conserved chaperones were shown to be involved in the assembly and disassembly of intracellular proteins and were located in the ER, nucleus and cytoplasm of cells. 23 From primitive single-cell organisms to sophisticated human immune cells dealing with inflammation, chaperones are essential for protein folding. In humans, there are ~194 chaperones expressed and in whole blood 122 chaperones have been detected at ≥10 transcripts per million. Some core chaperones are ubiquitous, while others are associated with tissue-specific functional networks. Many chaperones interact with other chaperones, which allows them to alter their function 24 and as neurologists are acutely aware, when some chaperones in brain cells decline with age, cellular protein aggregates increase. 25 Chaperones have evolved to be more versatile as they evolve from unicellular to multicellular organisms. The Escherichia coli chaperones are all constitutive but can be induced further upon environmental stress conditions. By contrast, human cells retain constitutive inducible chaperones; core chaperones conserved in all tissues ('essential house-keeping functions, e.g. UPR'), but others that can be differentially expressed across tissues (e.g. brain, lung, or muscle), giving chaperone a greater dynamic functionality in certain organs and cell types. 24 CRT is a very ancient protein present in all flora and fauna except, yeasts or bacteria, suggesting it has evolved its biological functions over 350 million years. 26 GRP78/BiP may be 400 million years old, as a homologue of this gene is present in yeast as KAR2 (Saccharomyces cerevisiae) and DnaK in bacteria, where its suppression leads to the inhibition of translocation of secretory proteins. 27 Currently, ER chaperones (e.g. CRT, calnexin, protein disulphide isomerase [PDI] and GRP78/BiP) are known to have pivotal roles in protein folding, cell survival and development. Often, these functions require that chaperones be retained within the ER. To facilitate, retention, the C-terminal KDEL amino acid sequence on chaperones, ensures that they are retained or recycled back into the ER. 28 It is becoming apparent that the location of various chaperones influences their function. For example, intracellular CRT is essential for glycoprotein folding, 29 but when transported to the surface of tumour cells can act as an 'eat-me' signal on pre-apoptotic cells, 30 or inhibit complement activation. 31 Once in the extracellular environment, it influences cellular apoptosis of immune cells 32 and phagocytosis 33,34 or binds to stimulatory co-factors such as LPS, which can influence immune regulation and inflammatory pathways as well as innate immunity, in multiple species. [35][36][37] Similarly, GRP78/BiP has an essential role to play in the quality assurance of protein assembly and transport from the ER but can also translocate to the cell membrane of stressed cells where it can engage in anti-and pro-survival functions. However, in an extracellular environment, it selectively binds and is rapidly internalized by myeloid cells-monocytes, macrophages and DCs where it can trigger a series of signalling pathways that dampen inflammation and restore immune homeostasis. 3-7,38

| How chaperones are naturally released from cells
Proteins destined for secretion are synthesized by ribosomes and translocate to the ER. The newly synthesized proteins interact with ER chaperones; GRP78/BiP, calnexin, CRT and PDI. These chaperones aid the assembly of polypeptides into mature proteins and assist their transport initially to the ER-Golgi intermediate compartment (IC). This processing and transportation of proteins are aided by chaperones such as CRT and GRP78/BiP, before the chaperones are returned to the ER via COPI vesicles, while the mature protein continues to the cell surface for insertion into the plasma membrane or secretion via the COPII vesicles (see Figure 2A). Although CRT and GRP78/BiP are chaperones with C-terminal KDEL sequences, their mode of release from cells differs (see below). Upon certain types of cellular stress, some chaperones actively get released from the cells along with some of their binding partners (see below). Once released from cells, we know much less about how specific chaperones function outside of cells. We are interested in how ER chaperones influence the innate and adaptive immune system once in the extracellular environment.

| Release of CRT from human immune cells-First observations
Interest in the release of endogenous CRT has gained momentum given its recent identified roles in immunogenic cell death of tumours 39 and myeloproliferative neoplasms (MNPs) 40 Numerous studies have investigated how CRT is released from cells during cell stress, pharmacological intervention or due to somatic mutations. 41 In 1994, Eggleton and colleagues observed CRT on the surface of neutrophils; stimulation with the bacterial tri-peptide FMLP led to the release of CRT into the extracellular medium where it is was shown to bind to innate immune molecules C1q and collectins. 42 Concurrently, a molecule on the surface of the monocytic cell line U937 which also bound to C1q and was named a 'C1q receptor' having the same sequence as CRT was identified. 43 These unexpected observations of an apparent ER chaperone interacting with a complement protein were intriguing. Further work showed that CRT could bind to C1q and block 'classical' complement activation. 31 Meanwhile, Pritchard and coworkers also observed CRT-like molecule on the surface of a persistent hookworm parasite-Necator americanus collected from Papua New Guinea. 44 They hypothesized and later confirmed that the presence of CRT on the surface of the parasite evolved as a mechanism by which hookworms avoid attack by the host's complement system. 45 More recently, work by Arturo Ferreira and his team have demonstrated that other parasites, particularly Tryanosoma cruzi, also present CRT on their cell surface to evade attack by the complement system. 46 This early work provoked an interest to study CRT-immune interactions further but did not explain how CRT cell surface expression was regulated either in humans or more primitive species.

| Extracellular CRT release is associated with autoimmunity
A link between immune cells, surface CRT and complement attracted interest when it became evident that dead and dying cells in the circulation were being cleared in a noninflammatory manner by a process of apoptosis and that changes in CRT expression were associated with this process. 47 It was shown that complement proteins were important in clearing apoptotic cells from the circulation, because a rare deficiency in which individuals lacked C1q led to the development of a severe autoimmune disease, systemic lupus erythematosus (SLE). Meanwhile, Walport and colleagues showed that C1q deficiency led to defective apoptosis in these patients. 48 It was proposed that extracellular CRT acted as an intermediary, binding F I G U R E 2 Exit of GRP78/BiP and entry of IRL201805 influences different cell signalling and functional pathways. (A) Under physiological conditions, the pH and Ca 2+ -regulated endoplasmic reticulum (ER) serves as a location for nascent peptides to interact with various KDELretaining chaperones where they undergo folding and insertion into COPII vesicles for transportation to the Golgi and secreted or inserted within the plasma membrane. During ER/oxidative stress, GRP78/BiP and KDEL-containing chaperones (e.g. BiP or CRT) can aid protein transport to the Golgi as part of the integrated stress response (ISR) and are retained by the KDEL receptors in the acidic conditions and then returned to the ER in COPI vesicles by retrograde translocation. (B) Under severe stress, the KDEL-containing chaperones bind to accumulated protein aggregates and travel with them from the ER to the Golgi. Here, the KDEL receptors become saturated and via this anterograde process, chaperones including BiP or CRT and aggregated proteins, are either retained in cytosolic inclusion bodies, or translocated to the cell surface where they bind to cell surface proteins/receptors or are secreted. (C) By contrast, the therapeutic exposure of cells to extracellular IRL201805 does not signal through the ER-Golgi KDEL-receptor pathways. It is rapidly internalized <2 h possibly by receptor-mediated endocytosis. Once in APCs, IRL201805 has direct tolerogenic effects on the cells, such as increase IDO, decrease pro-inflammatory cytokines, while increasing anti-inflammatory cytokines. There is also evidence that endogenous GRP78/BiP self-peptides are loaded in HLADR-II molecules and presented on the cell surface, where they may activate tolerogenic Tregs against self-peptides (see Section 3.5).
to C1q on the surface of apoptotic cells, while also docking with CD91 on professional phagocytes, thereby promoting uptake of cell debris by micropinocytosis. 49 Some former clues as to how CRT is released from cells came from the observations of Sontheimer, who observed that CRT transcriptional activity increased when dermal epithelial cells were stressed by a calcium ionophore, heat shock or heavy metals such as zinc and cadmium. 50 Sontheimer proposed that CRT was a HSP and, like GRP78/BiP, was overexpressed under certain cell stress conditions. Similarly to GRP/BiP, extracellular CRT could influence a number of immune pathways, for example inhibit complement activation and, 31 alter DC recognition and uptake of tumour cell antigens. 51 More recently, mutant forms of extracellular CRT have been shown to bind to a thrombopoietin (TPO) receptor, precipitating the induction of rare blood cancers called MPNs, in which stem cells in the bone marrow make excessive numbers of various blood cells. 52 In SLE, anti-CRT antibodies are detected and serum CRT protein levels are associated with disease activity, particularly nephritis damage 53 and RA. 16 In addition, citrullinated CRT, which is overabundant in the RA synovium, potentiates HLA-DRB1 share epitope signalling leading to increase bone erosion, PAD activation and raised TNF-alpha serum levels in experimental models of RA. 54,55

| Somatic mutant CRT is associated with myeloproliferative disorders
CRT, like GRP78/BiP, normally has a negatively charged C-terminal KDEL sequence (see Section 3.2), which aids CRT docking into the positively charged binding cavity of the KDEL receptor. 56 These biophysical features of CRT are an important aspect of the KDELR chaperone retention system to retain chaperones internally. This has been underscored in the last decade or so with human MPNs, where the frameshift in mutant CRT's produced, results in mutant proteins with positively charged C-termini. The MPNs are a rare group of blood cancers originating in the bone marrow in which the body makes too many of a particular type of blood cells. Somatic changes in CRT were originally observed in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF), which are both MPNs. 41,57 In these neoplasms, ~30-50 mutations have been found in CRT. Two common mutations consist of a 52 amino acid deletion (type I mutation) or a five amino acid insertion (type II mutation) in the C-terminus. In all cases, the ER retrieval signal (KDEL) is lost in the C-terminus region of the protein allowing these mutants to escape ER retention. 40 In a study of MPN patients (n = 113) carrying the mutant CRT, the KDEL-lacking mutant CRT was secreted into their plasma, with a mean concentration of ~24.6 ng/ml (range 0-156.5 ng/ml). 58 The mutations can lead to structural changes of the protein within the ER. Two positively charged C-domains have been shown to form a homodimer and a hypothetical model proposed, in which the homodimers may intertwine to form a dimeric complex, which facilitates the N-domains to bind to N-glycans on the thrombopoietin (TPO/MPL) receptor. 58,59 Normally, the P-domain of the protein can prevent this interaction. 60  it was proposed for surface CRT to provoke an immune response on tumour cells; it had to be released on pre-apoptotic cells. Not surprisingly, human cells in a necrotic state or in the early stages of apoptosis due to ER-Ca 2+ -dysregulation also present CRT on their cell surface or release CRT into the extracellular milieu. 33,67 To this end, the term immunogenic cell death (ICD) is used to distinguish the immune recognition function of surface CRT on pre-apoptotic cells versus CRT was found on the surface or released from necrotic or apoptotic cells. It is fortuitous that chemotherapeutic drugs such as anthracycline antibiotics and platinum-based platins act on many metabolic pathways in cells, including ER stress. Like many chemotherapeutic drugs, they are relatively toxic as such, they trigger ER cell stress particularly in rapidly proliferating tumour cells. This leads to ER stress proteins being activated, including CRT. A series of signalling events occur, resulting in phosphorylation of the eukaryotic translation initiation factor eIF2α by the PKR-like ER kinase (PERK), followed by proteolytic cleavage of ER-sessile protein BAP31 by caspase-8, and activation of proapoptotic proteins BAX and BAK.
This leads to an anterograde release of CRT from the ER to the Golgi apparatus and exocytosis of CRT-containing vesicles to the plasma membrane. 66 Vesicle-bound SNARE proteins that facilitate vesicle fusion aid this translocation. The notion that CRT translocates to the plasma membrane on its own is unlikely, as it is often associated with other proteins, for example ERp57. 64,68 Interestingly, other more physical cancer cell therapies, such as photodynamic treatments (PDT), can similarly lead to CRT appearing on the cell surface but do so independently of eIF2α phosphorylation or association of ERp57. This suggests there are numerous heterogenous vesicular transport pathways that may aid CRT presentation on the surface of pre-apoptotic cells.
Cell surface-bound CRT as opposed to extracellular CRT is considered the dominant ICD signalling molecule on tumour cells. Cell surface CRT is believed to disturb the balance between the CD47 ('don't eat me') signals with SIRPα on phagocytes, especially DCs.
The exon-9 mutated CRT can be released via the anterograde 'Golgi-secretory pathway'. 69 Alternatively, CRT can be released from necrotic cells in various pathological conditions (Table 1) or after chemotherapy. In addition, professional phagocytes such as monocyte/macrophages, that scrutinize and eliminate tumour cells, are known to release CRT via activation of Bruton's tyrosine kinase (BKT/TLRs) pathway. 70 CRT phosphorylation by BKT in macrophages is important for CRT trafficking to the cell surface to function as a bridging molecule as part of the CRT/CD91/C1q complex, which initiates phagocytosis of apoptotic cells. 71 Some patients with tumours are nonresponders to chemotherapeutic treatments that normally induce transport of CRT onto their tumour cell surface. Lin et al. 72 have studied cancer patients who do not respond to checkpoint inhibitors and revealed that they possess high expression of the gene coding for stanniocalcin-1 (STC1) that is associated with poor survival. They demonstrated that intracellular STC1 binds cytosolic CRT keeping it near to mitochondria and preventing CRT translocation to the cell surface.

| E X TR ACELLUL AR G RP78/B IP/18 05
Endoplasmic reticulum chaperones such as CRT and BiP are both capable of inducing adaptive immune responses once they are released from cells. There are, however, notable differences in how these two chaperones function. CRT promotes maturation of APCs, cross-presentation of tumour antigens and facilitates recruitment of Teffs, primarily CD8 cytotoxic T cells, that provide a robust immune response to tumours ( Figure 4B). For CRT, to drive this anticancer immunity, it requires release and membrane binding of endogenous CRT from pre-apoptotic cancer cells. By contrast, pharmacologically manufactured homologues of BiP, for example IRL201805 (Table S1) but, not release of endogenous BiP, promotes induction of tolerogenic APCs and promotes suppressive features of T-regulatory cells ( Figure 4A). The pharmacokinetics (PK) and pharmacodynamics (PD) also differ. Cell surface CRT is retained on the surface of tumour cells for days, until recognized by antitumour cells, and then, the cells are eliminated, representing a long PK, short PD. When IRL201805 is administered in vivo, it has a short serum half-life (1-4 h), but long PD > 12 weeks. Consequently, while cancer immunologists have focused on ways to manipulate release of endogenous CRT from cells, immunologists interested in preventing autoimmunity and resolving inflammation have focused on delivering extracellular IRL201805 to immune cells, for internalization and activation of immunosuppressive features of myeloid and T cells. For both these proteins, a comprehensive knowledge of how they respond with cells under resting and stress conditions is important, as this will aid our understanding of how long-term immune responses are triggered during stress conditions (e.g. tumour production or autoimmunity) before re-establishing a homeostatic rebalance upon alleviation of pathological conditions.

| Extracellular GRP78/BiP interaction with immune cells-First observations
In the 1990s, the discovery of elusive initiating autoantigens in RA was a focus for research. In a proteomics approach, denaturing polyacrylamide gel electrophoresis and immunoblotted were used to separate soluble proteins from human chondrocyte lysates. These The release of GRP78/BiP from cells in a number of diseases (Table 1) may be taken up by APCs, presented to T cells, allowing autoreactive T-regulatory cells (Tregs) and T-effector cells (Teffs) to be exposed to this self-antigen. 74 This might explain the antibody generation against GRP78/BiP observed too (Table 2). 38 However, in chronic diseases, low-dose exposure of self-antigens such as GRP78/BiP is a mechanism by which peripheral tolerance is established by downregulating the pro-inflammatory cells followed by autoreactive Treg production. 3 Therefore, identifying diseases where endogenous GRP78/BiP is secreted above a certain threshold might provide an opportunity where a single or series of high therapeutic doses of IRL201805 could accelerate a sustained immune tolerance response. Interestingly, several diseases have been recorded in which extracellular GRP78/BiP is present in plasma at higher levels than age-sex match control subjects (Table 1).

| GRP78/BiP release from cells via stress
GRP78/BiP, like several ER chaperone proteins, has a KDEL sequence that can engage with KDEL receptors. KDEL receptor 1 (KDELR) was originally found to be responsible for the return of soluble ER-resident proteins to the ER from the IC of the cis-Golgi. This retrograde transport requires soluble ER-resident proteins to either have a KDEL-like motif at their C-terminus or to form a complex with ER-resident proteins that do. 75 In the epithelial HeLa cell line, it has been reported that KDELR modulates ER stress responses. 76 More recent studies have suggested that KDELR function goes beyond motif recognition by demonstrating that the chaperonebound KDELR triggers the activation of Src family kinases at the Golgi complex, a phenomenon that may be critical for intracellular signalling cascades. 77 92 Once on the cell surface, GRP78/BiP has been reported to bind to numerous self-antigens and microbial proteins and consequently has been proposed as a nonspecific 'receptor' for a multitude of proteins. 93 Collectively, these independent studies reveal that GRP78/BiP can shuttle out of the cell with binding partners and relocate to the cell surface, where it becomes embedded in the plasma membrane.

| Entry of extracellular GRP78/ BiP/1805 and other HSPs into APCs by receptormediated endocytosis
Evidence of HSP entry into murine monocytic (P388D1) and DC

| Internalization of GRP78/BiP immunomodulates both APC/B/T-cell immune responses
Myeloid cells (monocytes and DCs) predominantly bind and internalize BiP or IRL201805. There are subsets of B cells and T cells that IRL201805 has been demonstrated to bind to directly. 96 In the peripheral blood, IRL201805 is rapidly internalized by monocytes ( Figure 2C), where IRL201805 has been shown to have a direct effect on various phenotypical and metabolic functions of myeloid cells. Osteoclast generation by cultured human monocytes is inhibited by BiP ( Figure 3A) and bone resorption decreased as measured by lacunar formation on dentine slices. 5    In a separate study focused on Type 1 diabetes, citrullinated GRP78/BiP R510 containing peptides have been shown by Buitinga and co-workers 108 to bind to the shared epitope HLA-DRB1*04:01 molecules in postcytokine-treated human islets from type 1 diabetes patients, which were associated with the identification of higher CD4 + T-cell frequencies directed against citrullinated GRP78 (citGRP78) epitopes. In this study, several native and citrullinated GRP78/BiP peptides were identified as binding to the DRB1*0401.
In addition, autoantibodies against citCRP78/BiP were identified in a subset of patients, providing further evidence that APC-processed GRP78/BiP peptides evoked CD4+ T-cell and B-cell responses.  Figure 4B). For both these chaperones, the natural levels of plasma endogenous GRP78/BiP or the surface expression of CRT on cells is most likely suboptimal to provoke an optimal immune response.

F I G U R E 4
In human therapeutic trials, the way IRL201805 or calreticulin is presented to dendritic cells governs their downstream immune cell signalling. (A) With infusion of IRL201805, it is rapidly taken up mainly by myeloid cells, including dendritic cells (DCs), leading to sustained tolerogenic changes in the DCs and possibly presentation of IRL201805 peptides to 'primed' Tregs which can direct immunosuppressive responses against autoimmune effector cells. (B) Calreticulin is not infused directly, but endoplasmic reticulum (ER) stress-inducing drugs such as anthracyclines in combination with chemotherapeutics target tumour cells leading to anterograde translocation of wildtype CRT to the surface of ER stress cancer cells, which acts as an 'eat-me signal to immature DCs, which rapidly mature and signal to cytotoxic T cells to destroy the cancer cells.
Therapeutic interventions have shown that increased concentration of these proteins can have clinical benefit.

| Therapeutic evidence for extracellular for CRT
The therapeutic potential of CRT has been examined in a number of

| Therapeutic evidence for extracellular BiP/1805
Work from a number independent laboratories provides evidence that GRP78/BiP is a self-antigen that is rapidly taken up by APCs,  Figure 3B). This is of interest because it has been previously shown that RA Tregs have an inability to induce the activation of the tryptophan-degrading enzyme IDO and CTLA-4 production is reduced, possibly by increased methylation of the CTLA-4 promoter in RA Tregs. 120 These multiple changes in regulatory molecules in both myeloid and lymphocytic cells provide compelling evidence that IRL201805 acts to fundamentally reset immune responses.
In the RAGULA clinical trial, the responsiveness to IRL201805 treatment was most striking in patients who had higher levels of basal natural GRP78/BiP (Data not shown) in their circulation prior to IRL201805 infusion. Within 2 weeks of infusion, patients in this responsive group had significantly lower serum levels of C-reactive protein, vascular endothelial growth factor and interleukin (IL)-8 serum levels than those of the placebo group. 117 We hypothesize that administrating pharmacological levels of IRL201805 can promote a greater immunosuppressive activity in APCs (monocyte/macrophage, DCs), which, in turn, activate Tregs that are primed to respond to self-antigen (GRP78/BiP) aiding resolution of inflammation during autoimmunity (Corrrigall et al., paper in preparation). Once the inflammatory processes are resolved, the immune system would be expected to return to immune homeostasis via changes in stress, redox and metabolic signals, maintaining a balance between tolerance and immunogenicity. This approach differs from current therapeutics that tend to be immunosuppressive in nature (e.g. NSAIDs, steroids, biologics and JAK inhibitors).

Evidence of autoantibody generation
With the development of any therapeutic, natural or synthetic, the presence of antidrug antibodies and possible complications that may arise from their generation have to be assessed.
In relation to GRP78/BiP and CRT as naturally occurring intracellular proteins, they are somewhat protected from immune surveillance under normal physiological conditions. However, under certain pathological/stress conditions, autoantibodies against endogenous GRP78/BiP and CRT have been detected in several disease states compared with control subjects (see Table 2). The generation of anti-GRP78/BiP or anti-CRT antibodies may act as a biomarker of protein release from stress cells, but it should be remembered that the presence of autoantibodies is generally quite benign. Very few are disease-specific or even pathologic and the majority play more of a part in diagnosis than therapy. As people age, the presence of autoantibodies in serum increases in variety and quantity without much harm to the healthy person. This occurs because proteins are continually being encountered by the immune system. However, if they cross-react with part of an endogenous protein, it is possible that they will generate autoantibodies. Such antibodies would only be of concern if they neutralized the effects of cell surface CRT in tumour resolution or altered extracellular IRL201805 efficacy in resetting autoimmune diseases. This conjecture requires further examination.
In some cases, presentation of antigens to generate autoreactive immunosuppressive Tregs and generation of B cells that recognize self-antigens can be beneficial in preventing autoimmunity. 121 There is a fine balance between activating some lymphocyte subsets and inhibiting others. What is clear is that both membrane-bound CRT and the extracellular manufactured modified homologue of GRP78/ BiP -IRL201805 can provoke physiologically relevant immunemodulating effects in selected diseases.

| CON CLUDING REMARK S
It has become clear over a number of decades that the ~20,000 protein-encoding genes in the human body, depending on how they are transcribed and the splice variants produced and posttranslationally modifications that arise can ultimately generate ~70,000 proteins. 122 Not surprisingly, many proteins have been revealed to have more than one function. 123 Then, each cell could contain up to 42 million individual protein molecules. 124 Chaperones more than most proteins have to be adaptable; they must be able to function in different redox conditions and have effective ion buffering capacity, bind to and disengage from peptides, folded and unfolded protein, help glycosylate secretory proteins with other specialist quality control proteins and then shuttle between organelles and occasionally to the cell surface and beyond. 125,126 In each location, they appear to have a different function. Once outside cells, chaperones switch from protein folding proteins to DAMPs (e.g. CRT) or regulatory-associated molecular patterns-RAMPs (e.g. GRP78/BiP).
As new generations of high-tech 'omics-driven' therapeutics are developing, Biotech and Pharma industries strive to generate engineered cells as bespoke therapies for a multitude of diseases. By contrast, these resolution-promoting chaperones, for example CRT and IRL201805, may herald a new generation of biologics affecting multiple pathways common to inflammatory diseases and thereby unlock some of the multifaceted immune-regulatory qualities of these highly conserved proteins.

ACK N OWLED G EM ENT
We would like to thank all our colleagues in the Chaperone field over the years who have made significant contributions to the extracellular functions of CRT and GRP78/BiP in physiology and medicine.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest regarding the calreticulin research cited in this article. All authors are employees or consultants of Revolo Biotherapeutics Limited, https://revol obio.com.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data cited in this article are openly available in a public repository that issues datasets with DOIs and all information with DOIs are listed in the reference section of the article.